【摘要】：子癇前期(preeclampsia,PE)是懷孕期間特有的并發(fā)癥,特點是水腫、高血壓、尿蛋白并存,涉及多個器官系統(tǒng)損害。產(chǎn)后上述癥狀仍持續(xù)存在,它是導(dǎo)致全世界母嬰病死率增加的主要原因之一,發(fā)病率在全球孕婦中為5%~8%。盡管目前針對子癇前期的發(fā)病有多種學(xué)說,但其發(fā)病機(jī)制仍不明確。PE分為輕度和重度,重度子癇前期以34周為界分為兩個主要亞型,早發(fā)型(early-onset severe pre-eclampsia,EPE)與晚發(fā)型重度子癇前期(late-onset severe pre-eclampsia,LPE)。EPE的特點為妊娠早中期出現(xiàn)血壓及尿蛋白迅速升高,終末器官損害嚴(yán)重,并發(fā)癥較多,其胎盤功能損傷嚴(yán)重,因此又稱為“胎盤源性疾病”,胎兒在宮內(nèi)生長受限,母嬰結(jié)局的預(yù)后較差,已經(jīng)引起產(chǎn)科醫(yī)生高度重視;而LPE的病情發(fā)展緩慢,各種臨床癥狀與體征均較EPE輕,胎盤形態(tài)學(xué)基本正常,多與母體自身疾病相關(guān),又稱為“母源性疾病”。核轉(zhuǎn)錄因子5(nuclear factor of activated T cells 5,NFAT5)廣泛存在于眼睛、肝臟、腎臟、胎盤等多個器官表達(dá)。高滲條件下被激活,調(diào)節(jié)多種滲透性保護(hù)基因的表達(dá)。組織局部滲透壓升高與機(jī)體的炎癥狀態(tài)緊密相關(guān)。NFAT5在高滲條件下激活后能夠促進(jìn)炎癥因子的表達(dá),在自身免疫、細(xì)胞應(yīng)激、炎癥性疾病方面發(fā)揮著重要的作用。單核細(xì)胞趨化蛋白1(monocyte chemoattractant protein-1,MCP-1)含有76個氨基酸,與其受體(chemokine receptor 2,CCR2)結(jié)合后趨化或激活炎癥細(xì)胞聚集到炎癥部位,參與機(jī)體的病理生理反應(yīng)。有研究顯示滋養(yǎng)細(xì)胞和蛻膜細(xì)胞可分泌MCP-1,趨化血液中單核巨噬細(xì)胞遷移至蛻膜,在螺旋動脈重塑過程中意義重大。最近研究表明在腹膜間皮細(xì)胞Met5A細(xì)胞中MCP-1啟動子區(qū)域有Ton E結(jié)合位點,NFAT5與其結(jié)合后可導(dǎo)致MCP-1表達(dá)升高,然而,在子癇前期胎盤組織中NFAT5與MCP-1是否存在相關(guān)性國內(nèi)外未見報道,本研究以此為切入點,探討子癇前期患者胎盤組織中NFAT5與MCP-1之間的相關(guān)性,為子癇前期的發(fā)病機(jī)制開辟一個新窗口。巨噬細(xì)胞能夠清除體內(nèi)細(xì)胞凋亡成分,它的免疫調(diào)節(jié)作用在妊娠期子宮胎盤床附近受到關(guān)注。子癇前期患者中有大量的巨噬細(xì)胞浸潤,通過多種機(jī)制調(diào)控著滋養(yǎng)細(xì)胞的侵襲能力。目前,國外對于巨噬細(xì)胞與子癇前期的關(guān)系已做了很多研究,但是巨噬細(xì)胞浸潤和MCP-1、NFAT5的關(guān)系國內(nèi)外研究較少。本研究以此為突破口,通過分析巨噬細(xì)胞浸潤和MCP-1、NFAT5的關(guān)系,初步探討其在子癇前期發(fā)病機(jī)制中的作用。目的本研究通過比較MCP-1在不同類型重度子癇前期患者母血、臍血及胎盤組織中的水平,并分析其相關(guān)性,為將來早期診斷子癇前期孕婦提供分子標(biāo)記物和新靶點;通過檢測不同類型重度子癇前期胎盤中MCP-1、NFAT5及CD68+巨噬細(xì)胞的表達(dá),分析巨噬細(xì)胞浸潤和MCP-1、NFAT5的關(guān)系,初步探討其在子癇前期發(fā)病機(jī)制中的作用。方法1研究對象選取在鄭州大學(xué)第三附屬醫(yī)院產(chǎn)科住院分娩的重度子癇前期患者61例為試驗組,其中EPE 31例(早發(fā)組),LPE 30例(晚發(fā)組),均為剖宮產(chǎn)分娩,收集標(biāo)本時間為2015年1月至2016年1月。另選取同期正常孕婦30例為對照組,均為健康足月孕婦,因社會因素或骨盆異常等原因行剖宮產(chǎn)。各組孕婦均為單胎妊娠,此次妊娠均為自然受孕,未采取輔助生殖技術(shù);各組孕婦既往均無自身免疫性疾病、糖尿病、染色體結(jié)構(gòu)異常以及胎膜早破等其他妊娠合并癥病史。2實驗方法1.血清及胎盤標(biāo)本采集:所有研究對象入院后次日清晨空腹抽取孕婦肘靜脈血3ml,臍靜脈血則是在胎盤娩出后抽取3ml。室溫下放置半小時,隨后離心約15min,取上層淡黃色血清分裝在EP管中,避免溶血標(biāo)本,置于-80℃冰箱保存待測。所取的胎盤組織標(biāo)本均在剖宮產(chǎn)胎盤娩出后15 min內(nèi),在母體面中央?yún)^(qū)域(無出血、梗死及鈣化),剪下大小約(1.0 cm×1.0 cm×1.0 cm)2塊組織,用生理鹽水漂洗后,其中一塊投入液氮,后轉(zhuǎn)-80℃冰箱保存?zhèn)溆�。另一塊固定于福爾馬林溶液中,用于免疫組化實驗。2.采用酶聯(lián)免疫吸附(enzyme-linked immunoadsordent assay,ELISA)方法對孕婦母血清、臍血清中MCP-1的水平進(jìn)行檢測。3.采用Real-time PCR技術(shù)檢測胎盤組織中NFAT5和MCP-1 m RNA的表達(dá)水平,數(shù)據(jù)統(tǒng)計分析采用2-△△CT相對定量方法。4.采用免疫組化方法檢測NFAT5、MCP-1、CD68+巨噬細(xì)胞在胎盤中的表達(dá)。通過圖像分析技術(shù)對上述三個指標(biāo)的免疫組化結(jié)果進(jìn)行分析比較。3統(tǒng)計學(xué)處理統(tǒng)計學(xué)分析采用SPSS 21.0軟件。頻數(shù)資料采用(?)±s表示,多組之間的比較采用單因素方差分析進(jìn)行,多組之間的兩兩相比用LSD-t法;用Pearson方法分析兩組之間的相關(guān)性,檢驗水準(zhǔn)為α=0.05。結(jié)果1三組間一般臨床資料的比較對照組、早發(fā)組和晚發(fā)組孕婦之間的年齡、終止孕周、分娩前BMI比較,差異均無統(tǒng)計學(xué)意義(P0.05);早發(fā)組新生兒出生體重、胎盤重量均低于對照組和晚發(fā)組,差異均有統(tǒng)計學(xué)意義(P0.05);早發(fā)組24h尿蛋白高于晚發(fā)組,差異有統(tǒng)計學(xué)意義(P0.05)。2三組間母血清、臍血清中MCP-1水平的比較早發(fā)組母血清中MCP-1水平185.80±15.73ng/L高于晚發(fā)組164.93±6.24ng/L和對照組154.78±9.61ng/L(P0.05);早發(fā)組臍血清中MCP-1水平為223.49±41.68ng/L高于晚發(fā)組151.85±13.92ng/L和對照組113.91±6.67ng/L(P0.05)。3三組間胎盤中NFAT5和MCP-1 m RNA表達(dá)水平的比較早發(fā)組胎盤中NFAT5 m RNA的表達(dá)水平為(1.07±0.03),高于晚發(fā)組(0.92±0.02)及對照組(0.90±0.04)(P0.05);早發(fā)組胎盤中MCP-1 m RNA的表達(dá)水平為(1.08±0.12)高于晚發(fā)組(1.02±0.08)及對照組(0.99±0.02)(P0.05)。4三組間胎盤中NFAT5、MCP-1、CD68+巨噬細(xì)胞蛋白水平表達(dá)的比較早發(fā)組胎盤中NFAT5蛋白的表達(dá)水平為(34.16±4.65),高于晚發(fā)組(11.39±3.77)及對照組(10.65±1.85)(P0.05);早發(fā)組胎盤中MCP-1蛋白的表達(dá)水平為(38.47±4.81)高于晚發(fā)組(19.29±3.86)及對照組(20.33±3.92)(P0.05);早發(fā)組胎盤中CD68+巨噬細(xì)胞的表達(dá)水平為(80.60±10.47)高于晚發(fā)組(24.25±6.69)及對照組(22.79±5.58)(P0.05)。5 NFAT5、MCP-1與CD68+巨噬細(xì)胞浸潤的相關(guān)性分析1.早發(fā)組臍血清與母血清中MCP-1呈正相關(guān)(r=0.587,P0.05);早發(fā)組臍血清與胎盤中MCP-1呈正相關(guān)(r=0.754,P0.05);而在晚發(fā)組中也均呈正相關(guān)(r=0.576,0.791,P均0.05);早發(fā)組及晚發(fā)組臍血清中MCP-1與新生兒出生體質(zhì)量均呈負(fù)相關(guān)(r=-0.508,-0.496,P均0.05)。2.早發(fā)組及晚發(fā)組母血清中MCP-1水平與胎盤中MCP-1 m RNA的表達(dá)水平均呈正相關(guān)(r=0.671,0.676,P均0.05)。3.早發(fā)組胎盤中NFAT5 m RNA與MCP-1 m RNA表達(dá)水平呈正相關(guān)(r=0.724,P0.05),早發(fā)組胎盤中NFAT5 m RNA表達(dá)水平與CD68+巨噬細(xì)胞的表達(dá)呈正相關(guān)(r=0.640,P0.05);而在晚發(fā)組中也均呈正相關(guān)(r=0.631,0.608,P均0.05)。4.早發(fā)組及晚發(fā)組胎盤中MCP-1 m RNA表達(dá)水平與CD68+巨噬細(xì)胞的表達(dá)均呈正相關(guān)(r=0.704,0.686,P均0.05)。結(jié)論1.MCP-1在重度子癇前期孕婦母血、臍血和胎盤中具有相關(guān)性,檢測其水平為早發(fā)型子癇前期的早期診斷提供依據(jù)。2.早發(fā)型重度子癇前期胎盤中NFAT5、MCP-1、CD68+巨噬細(xì)胞的表達(dá)兩兩間呈正相關(guān),說明NFAT5-MCP-1-CD68+巨噬細(xì)胞途徑可能在早發(fā)型重度子癇前期的發(fā)病過程中發(fā)揮作用。
[Abstract]:Preeclampsia (PE) is a special complication during pregnancy, characterized by edema, high blood pressure, and urinary protein coexistence, involving multiple organ system damage. The postpartum symptoms continue to exist, which is one of the main causes of the increase of maternal and infant mortality in the world, the incidence of 5%~8%. in pregnant women around the world, although the current needle before the eclampsia There are a variety of doctrines in the pathogenesis, but the pathogenesis is still not clearly defined as mild and severe.PE, and severe preeclampsia is divided into two major subtypes with 34 weeks as the boundary. Early onset (early-onset severe pre-eclampsia, EPE) and late onset severe preeclampsia (late-onset severe pre-eclampsia, LPE).EPE are characterized by early and mid trimester blood pressure. And the rapid increase in urine protein, serious end organ damage, more complications and serious injury of the placenta function, so it is also called "placental source disease", the fetus is limited in intrauterine growth, the prognosis of the mother and baby is poor, and the obstetricians have paid much attention to it, and the disease of LPE develops slowly, and all kinds of clinical symptoms and signs are lighter than EPE. The disc morphology is basically normal and is related to the mother's own disease. It is also called "maternal disease". Nuclear transcription factor 5 (nuclear factor of activated T cells 5, NFAT5) is widely expressed in many organs such as eyes, liver, kidney, and placenta. Under hypertonic conditions, it is activated to regulate the expression of various osmotic protective genes. Tissue local osmosis Elevated pressure is closely related to the inflammatory state of the body..NFAT5 can promote the expression of inflammatory factors after activation in hypertonic conditions. It plays an important role in autoimmune, cell stress, and inflammatory diseases. Monocyte chemoattractant protein 1 (monocyte chemoattractant protein-1, MCP-1) contains 76 amino acids, and its receptor (chemokine R). Eceptor 2, CCR2) combine to chemotaxis or activate inflammatory cells to accumulate to the site of inflammation and participate in the body's pathophysiological response. Studies have shown that trophoblast and decidual cells can secrete MCP-1 and migrate to the decidua by mononuclear macrophages in the chemotaxis, which is significant in the process of spiral artery remodeling. Recent studies have shown that Met5 in peritoneal mesothelial cells There is a Ton E binding site in the MCP-1 promoter region of A cells. NFAT5 and its binding can lead to the increase of MCP-1 expression. However, there is no correlation between the existence of NFAT5 and MCP-1 in the placental tissue of preeclampsia. This study is the breakthrough point to explore the correlation between NFAT5 and MCP-1 in placental tissues of preeclampsia patients, which is eclampsia. The early pathogenesis opens up a new window. Macrophages can remove the body's apoptotic components, its immune regulation is concerned near the pregnancy uterus placenta bed. A large number of macrophages infiltrate in preeclampsia, and regulate the invasion ability of the nourishing cell through a variety of mechanisms. A lot of studies have been done on the relationship between cell and preeclampsia, but the relationship between macrophage infiltration and MCP-1, NFAT5 is seldom studied at home and abroad. This study is a breakthrough. By analyzing the relationship between macrophage infiltration and MCP-1 and NFAT5, the role of the macrophage infiltration in the pathogenesis of preeclampsia was preliminarily discussed. The purpose of this study was to compare MCP-1 in different classes. The level of maternal blood, umbilical blood and placenta tissue in patients with severe preeclampsia and their correlation were analyzed to provide molecular markers and new targets for the early diagnosis of preeclampsia. By detecting the expression of MCP-1, NFAT5 and CD68+ macrophages in different types of severe preeclampsia, the infiltration of macrophages and the correlation of MCP-1 and NFAT5 were analyzed. A preliminary study of its role in the pathogenesis of preeclampsia. Method 1 the subjects selected 61 cases of severe preeclampsia in the obstetric department of the Third Affiliated Hospital of Zhengzhou University, 61 cases of severe preeclampsia, of which 31 cases (early onset group) and 30 LPE (late onset group) were Caesarean birth, and the collection time was from January 2015 to January 2016. At the same time, 30 normal pregnant women were selected as the control group, all of which were healthy full term pregnant women. All pregnant women were single pregnancy due to social factors or pelvic abnormality. All pregnant women were single pregnancy. All pregnant women were pregnant with natural pregnancy and did not adopt auxiliary reproductive technology. .2 experimental methods of other pregnancy complications, such as premature rupture of membrane, 1. serum and placenta samples: all the subjects were taken on the next morning after admission to the pregnant women's elbow vein blood 3ml. The umbilical vein blood was placed at the room temperature of 3ml. for half an hour after the delivery of the placenta, and then centrifuged for about 15min, and the upper layer of light yellow sera was separated into the EP tube to avoid dissolving. The specimen of the blood was kept in the refrigerator at -80 C for 15 min after cesarean section. In the central region of the mother body (no bleeding, infarction and calcification), 2 tissues were cut down (1 cm x 1 cm x 1 cm), and one of them was washed with saline, and the other was stored in the refrigerator at -80 centigrade. The block was fixed in the formalin solution and used in the immuno histochemical experiment.2. using enzyme-linked immunoadsordent assay (ELISA) method to detect the level of MCP-1 in maternal maternal serum and umbilical serum..3. was used to detect the expression level of NFAT5 and MCP-1 m in placenta tissue by Real-time PCR technique. Data statistical analysis was adopted. 2- Delta Delta CT relative quantitative method.4. was used to detect the expression of NFAT5, MCP-1, CD68+ macrophages in the placenta by immunohistochemical method. The immunohistochemical results of the above three indexes were analyzed by image analysis technique and compared with.3 statistics processing statistics analysis using SPSS 21 software. Frequency data were expressed by (?) + s, and the ratio of multiple groups was compared. Compared with the single factor analysis of variance, 22 of the multiple groups were compared with the LSD-t method, and the correlation between the two groups was analyzed with the Pearson method. The test level was the comparison group of the general clinical data between the 1 three groups of the alpha =0.05. results, the age, the final gestational age and the BMI comparison between the pregnant women in the early onset group and the late onset group were not statistically significant. P0.05. The birth weight of early onset group was lower than that of the control group and late onset group. The difference was statistically significant (P0.05). The 24h urine protein in the early onset group was higher than that in the late onset group (P0.05), the difference was statistically significant (P0.05) of the mother serum of.2 three groups, and the level of MCP-1 in the umbilical serum was compared to the level of MCP-1 in the maternal serum of the early onset group, and the level of MCP-1 was higher than that in the late onset group. The group 164.93 + 6.24ng/L and the control group were 154.78 + 9.61ng/L (P0.05), and the level of MCP-1 in the umbilical serum in the early onset group was 223.49 + 41.68ng/L higher than that in the late onset group 151.85 + 13.92ng/L and the control group was 113.91 + 6.67ng/L (P0.05).3 three. The expression level of NFAT5 and MCP-1 m in the placenta was (1.07 + 0.03). Higher than the late onset group (0.92 + 0.02) and the control group (0.90 + 0.04) (P0.05), the expression level of MCP-1 m RNA in the placenta of the early onset group was higher than that in the late onset group (1.02 + 0.08) and the expression of NFAT5, MCP-1, and CD68+ macrophage protein in the placenta of the control group (0.99 + 0.02) (0.99 + 0.02) (P0.05).4 three. The expression level of NFAT5 protein in the placenta of the early onset group (34.16 + 4.65), higher than that in the late onset group (11.39 + 3.77) and the control group (10.65 + 1.85) (P0.05), the expression level of MCP-1 protein in the placenta of the early onset group was higher than that in the late onset group (19.29 + 3.86) and the control group (20.33 + 3.92) (P0.05), and the expression level of the placenta in the early onset group was higher than that of the late onset group. The correlation between (22.79 + 5.58) (22.79 + 5.58) (P0.05).5 NFAT5, MCP-1 and CD68+ macrophage infiltration in the early onset group, the umbilical serum was positively correlated with the MCP-1 in the maternal serum (r=0.587, P0.05); the umbilical serum in the early onset group was positively correlated with the MCP-1 in the placenta (r=0.754, P0.05), but also in the late onset group (r=0.576,0.791, 0.05), and the early onset and late onset groups There was a negative correlation between MCP-1 in umbilical serum and the quality of newborn birth body (r=-0.508, -0.496, P 0.05). The level of MCP-1 in the early hair group and the maternal serum of the late onset group was positively correlated with the expression level of MCP-1 m RNA in the placenta (r=0.671,0.676, P are 0.05). The expression level of NFAT5 m RNA in the placenta of hair group was positively correlated with the expression of CD68+ macrophage (r=0.640, P0.05), but also in the late onset group (r=0.631,0.608, P all 0.05).4. early onset group and the expression level of MCP-1 m RNA in the late hair group was positively correlated with the expression of macrophage. The correlation between maternal blood, umbilical cord blood and placenta in preeclampsia pregnant women was found to provide a basis for early diagnosis of early onset preeclampsia to provide a basis for 22 positive correlations between the expression of NFAT5, MCP-1, and CD68+ macrophages in the placenta of early onset severe preeclampsia, indicating that the NFAT5-MCP-1-CD68+ macrophage pathway may be in early onset severe eclampsia. It plays a role in the process of early onset.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R714.244

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2 劉全;賀晶;董e，

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